Identification of an Agonist-induced Conformational Change Occurring Adjacent to the Ligand-binding Pocket of the M3 Muscarinic Acetylcholine Receptor

Much evidence indicates that, during activation of seven-transmembrane (7TM) receptors, the intracellular segments of the transmembrane helices (TMs) move apart with large amplitude, rigid body movements of especially TM-VI and TM-VII. In this study, AspIII:08 (Asp 113 ), the anchor point for monoamine binding in TM-III, was used as the starting point to engineer activating metal ion sites between the extracellular segments of the ␤ 2 -adrenergic receptor. Cu(II) and Zn(II) alone and in complex with aromatic chelators acted as potent (EC 50 decreased to 0.5 M) and efficacious agonists in sites constructed between positions III:08 (Asp or His), VI:16 (preferentially Cys), and/or VII:06 (preferentially Cys). In molecular models built over the backbone conformation of the inactive rhodopsin structure, the heavy atoms that coordinate the metal ion were located too far away from each other to form high affinity metal ion sites in both the bidentate and potential tridentate settings. This indicates that the residues involved in the main ligand-binding pocket will have to move closer to each other during receptor activation. On the basis of the distance constraints from these activating metal ion sites, we propose a global toggle switch mechanism for 7TM receptor activation in which inward movement of the extracellular segments of especially TM-VI and, to some extent, TM-VII is coupled to the well established outward movement of the intracellular segments of these helices. We suggest that the pivots for these vertical seesaw movements are the highly conserved proline bends of the involved helices. Seven-transmembrane (7TM)2 receptors, which couple through G proteins but also through a number of G protein-independent signaling pathways such as arrestin-mediated kinase activation, constitute the largest family of proteins in the human genome (1-3). These receptors are activated by basically all kinds of chemical messengers in the body, and they act as chemosensors for a multitude of external chemical signals. 7TM receptors are also the target for the majority of drugs, which, however, address only a small fraction of the receptor repertoire (4). Because they are involved as regulators of key physiological functions throughout the body, 7TM receptors are one of the major focus areas of the pharmaceutical industry (4, 5).7TM receptors are activated by agonists that span a chemical diversity range from large glycoprotein hormones over neuropeptides, lipid messengers, and nucleotides to small monoamines, amino acids, and even metal ions such as calcium (1-3). Notably, although there apparently is no common "lock" for all these chemically highly diverse "keys" (6), it is nevertheless expected that there is a common molecular activation mechanism for 7TM receptors as such. The conformational changes that accompany receptor activation have been characterized in rather great detail for the intracellular segments of the seven-helix bundle (7)(8)(9)(10)(11)(12)(13)(14). This has been achieved through a number of different biochemi...

Section: Global Toggle Switch Model For Helical Movements During 7tmsupporting

confidence: 76%

Metal Ion Site Engineering Indicates a Global Toggle Switch Model for Seven-transmembrane Receptor Activation

Elling

Frimurer²,

Gerlach

et al. 2006

“…Upon 7TM receptor activation, the top of TM-III and -VI move closer, and it has been speculated that an inverse agonist functions as a wedge preventing these helices from approaching each other (28,29). In agreement with this hypothesis, it has been shown that III:04/3.36 and VII:09/7.42 in fact move close together upon activation of the M3 muscarinic receptor (43). Thus, given that GSK682753A probably resides between these residues, it can be speculated that the compound inhibits the constitutive activity of EBI2 by preventing TM-III and -VII from approaching each other.…”

supporting

confidence: 57%

Ligand Modulation of the Epstein-Barr Virus-induced Seven-transmembrane Receptor EBI2

Benned-Jensen

Smethurst

Holst

et al. 2011

The Epstein-Barr virus-induced receptor 2 (EBI2) is a constitutively active seven-transmembrane receptor, which was recently shown to orchestrate the positioning of B cells in the follicle. To date, no ligands, endogenously or synthetic, have been identified that modulate EBI2 activity. Here we describe an inverse agonist, GSK682753A, which selectively inhibited the constitutive activity of EBI2 with high potency and efficacy. In cAMP-response element-binding protein-based reporter and guanosine 5-3-O-(thio)-triphosphate (GTP␥S) binding assays, the potency of this compound was 2.6 -53.6 nM, and its inhibitory efficacy was 75%. In addition, we show that EBI2 constitutively activated extracellular signal-regulated kinase (ERK) in a pertussis toxin-insensitive manner. Intriguingly, GSK682753A inhibited ERK phosphorylation, GTP␥S binding, and cAMP-response element-binding protein activation with similar potency. Overexpression of EBI2 profoundly potentiated antibody-stimulated ex vivo proliferation of murine B cells compared with WT cells, whereas this was equivalently reduced for EBI2-deficient B cells. Inhibition of EBI2 constitutive activity suppressed the proliferation in all cases. Importantly, the suppression was of much higher potency (32-fold) in WT or EBI2-overexpressing B cells compared with EBI2-deficient counterparts. Finally, we screened GSK682753A against an EBI2 mutant library to determine putative molecular binding determinants in EBI2. We identified Phe 111 at position III:08/3.32 as being crucial for GSK682753A inverse agonism because Ala substitution resulted in a >500-fold decrease in IC 50 . In conclusion, we present the first ligand targeting EBI2. In turn, this molecule provides a useful tool for further characterization of EBI2 as well as serving as a potent lead compound.The Epstein-Barr virus (EBV) induced receptor 2 (EBI2; also known as GPR183) is an orphan member of the 7TM 2 receptor family A. EBI2 is up-regulated up to 200-fold in B cells following EBV infection (1). In agreement with an expression pattern primarily restricted to immunological cell types (1, 2), it was recently demonstrated that EBI2 orchestrates the positioning of B cells in the follicle (3, 4). Specifically, expression of EBI2 allowed activated B cells to translocate to the outer follicular regions; in contrast, the absence of EBI2 permitted the cells to enter the germinal centers. Thus, EBI2 mediates the correct localization of B cells during humoral immune responses. To date, no endogenous ligand for EBI2 has been identified. Given that this receptor does not have a close homolog, the chemical nature of such a ligand is difficult to infer. Accordingly, EBI2 has been placed in varying 7TM receptor subgroups by different phylogenetic analyses as being a target of peptide (1, 5) or lipid ligands (6). Nevertheless, many aspects of EBI2 signaling have been revealed. Thus, we have previously demonstrated that EBI2 is constitutively active through G␣ i (but not G␣ q or G␣ s ) and serum response element (but not NFAT or NFB...

“…Cells were transfected with 4 g/dish of receptor plasmid DNA using the Lipofectamine Plus kit (Invitrogen), according to the manufacturer's instructions. To achieve higher receptor expression levels, transfected cells were incubated with 1 M atropine for the last 24 h of culture, as described previously (23)(24)(25)(26).…”

Section: Methodsmentioning

confidence: 99%

“…To monitor ligand-induced changes in receptor structure, we employed an in situ disulfide cross-linking strategy that allows the detection of disulfide bond formation between Cys residues that are adjacent to each other in the three-dimensional structure of the receptor (23)(24)(25)(26). One major advantage of this strategy is that ligand-dependent conformational changes can be detected in receptors present in their native membrane environment, without the need for any receptor purification and reconstitution steps.…”

mentioning

confidence: 99%

Distinct Structural Changes in a G Protein-coupled Receptor Caused by Different Classes of Agonist Ligands

Han

Hamdan

et al. 2007

Self Cite

The activity of G protein-coupled receptors can be modulated by different classes of ligands, including agonists that promote receptor signaling and inverse agonists that reduce basal receptor activity. The conformational changes in receptor structure induced by different agonist ligands are not well understood at present. In this study, we employed an in situ disulfide crosslinking strategy to monitor ligand-induced conformational changes in a series of cysteine-substituted mutant M 3 muscarinic acetylcholine receptors. The observed disulfide cross-linking patterns indicated that muscarinic agonists trigger a separation of the N-terminal segment of the cytoplasmic tail (helix 8) from the cytoplasmic end of transmembrane domain I. In contrast, inverse muscarinic agonists were found to increase the proximity between these two receptor regions. These findings provide a structural basis for the opposing biological effects of muscarinic agonists and inverse agonists. This study also provides the first piece of direct structural information as to how the conformations induced by these two functionally different classes of ligands differ at the molecular level. Given the high degree of structural homology found among most G protein-coupled receptors, our findings should be of broad general relevance.